Lipolytic enzyme variants

ABSTRACT

The substrate specificity of a lipolytic enzyme can be modified by making alterations to the amino acid sequence in a defined region of the lipolytic enzyme, so as to increase the level of a desired activity or to decrease the level of an undesired activity. Thus, the inventors have developed lipolytic enzyme variants with a modified amino acid sequence with a substrate specificity which can be tailored for specific uses.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 10/779,427, filed Nov. 22, 2004, now U.S. Pat. No. 7,312,062, which is a division of U.S. application Ser. No. 09/856,819, filed May 24, 2001, now abandoned, which claims priority from application serial no. PCT/DK99/00664, filed on Nov. 29, 1999, which claims the benefit of U.S. application Nos. 60/111,430, filed Dec. 8, 1998, 60/129,914 filed Apr. 19, 1999, and 60/160,735, filed Oct. 22, 1999, and clams priority under 35 U.S.C. 119 of Danish application serial nos. PA 1998 01572, filed Nov. 27, 1998, PA 1999 00391, filed Mar. 22, 1999, and PA 1999 01481, filed Oct. 15, 1999, the contents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a method of altering the substrate specificity of a lipolytic enzyme by modifying the amino acid sequence, and to lipolytic enzyme variants obtained by such modification. The invention also relates to a screening method for lipolytic enzymes.

BACKGROUND OF THE INVENTION

Lipolytic enzymes (such as lipases and phospholipases) are capable of hydrolyzing carboxylic ester bonds in a substrate to release carboxylic acids. The hydrolytic activity on different ester bonds is important for the usefulness of the lipolytic enzyme in various industrial applications.

Thus, enzymes with a high phospholipase activity are useful in a wide range of applications such as baking (U.S. Pat. No. 4,567,046), filtration of wheat starch hydrolysate (U.S. Pat. No. 5,264,367) and treatment of vegetable oil to reduce the content of phospholipid (U.S. Pat. No. 5,264,367). For the treatment of vegetable oil, the enzyme should have a low lipase activity, i.e. a low hydrolytic activity towards ester bonds in triglycerides.

WO 98/45453 indicates that an enzyme with a high hydrolytic activity on digalactosyl diglyceride (DGDG) is useful in baking.

It is well known to add a lipase to laundry detergents to aid in the removal of greasy soils (e.g. EP 258,068).

The release of short-chain fatty acids as free fatty acids (FFA) may be desirable for flavor development in food products, e.g. in cheese ripening (M. Hanson, ZFL, 41 (10), 664-666 (1990)).

The three-dimensional (3D) structure of several lipolytic enzymes is known, and several structures are known to contain a so-called “lid” which may be in an open or closed state covering the active site. Brady et al., Nature, 343, 767-770 (1990). Brzozowski A M et al., Nature, 351, 491 (1991). Derewenda et al., Biochemistry, 31 (5), 1532-1541 (1992).

F. Hara et al., JAOCS, 74 (9), 1129-32 (1997) indicates that some lipases have a certain phospholipase activity, whereas most lipases have little or no activity on phospholipids. Thus, phospholipase activity has been described in the lipases from guinea pig pancreas, Fusarium oxysporum and Staphylococcus hyicus, and attempts have been made to relate the phospholipase activity to the structure of the lipase. WO 98/26057; M. D. van Kampen et al., Chemistry and Physics of Lipids, 93 (1998), 39-45; A. Hjorth et al., Biochemistry 1993, 32, 4702-4707.

The prior art has described the effect on chain-length selectivity by amino acid substitutions in a lipase from Rhizopus delemar. Thus, R. D. Joerger et al., Lipids, 29 (6), 377-384 (1994) indicates that the variants F95D, F112W and V209W have an altered preference to C₄ and C₈ acids. R. R. Klein et al., JAOCS, 74 (11), 1401-1407 (1997) shows that the variant V206T+F95D has a higher selectivity for C₈ acid. R. R. Klein et al., Lipids, 32 (2), 123-130 (1997) indicates that the variants V209W+F112W, V94W and F95D+F214R have a higher hydrolytic activity towards C₄ and C₈ acids, and suggests that structural determinants for medium-chain length specificity may reside in the distal end of the acyl binding groove.

SUMMARY OF THE INVENTION

The inventors have found that the substrate specificity of a lipolytic enzyme can be modified by making alterations to the amino acid sequence in a defined region of the lipolytic enzyme, so as to increase the level of a desired activity or to decrease the level of an undesired activity. Thus, the inventors have developed lipolytic enzymes with a modified amino acid sequence (hereinafter called lipolytic enzyme variants, or variants for short) with a substrate specificity which can be tailored for specific uses.

Accordingly, the invention provides a method of producing a lipolytic enzyme variant and lipolytic enzyme variants prepared by the method. The method comprises:

a) selecting a substrate and an ester bond of interest,

b) selecting a parent lipolytic enzyme,

c) selecting at least one amino acid residue in a region near the active site, near the C-terminal or in the lid region of the parent lipolytic enzyme as described below,

d) making alterations each of which is an insertion, a deletion or a substitution of the amino acid residue,

e) optionally, making alterations each of which is an insertion, a deletion or a substitution of an amino acid residue at one or more positions other than c),

f) preparing the resulting variant,

g) testing the activity of the variant on the ester bond in the substrate, and

h) selecting a variant having an altered activity on the ester bond.

Thus, in one aspect, the parent lipolytic enzyme has an alcohol binding site having a glycerol part with an sn2 position, and the amino acid alteration is within 10 Å of the C atom at the sn2 position of the glycerol part of a substrate triglyceride.

In another aspect, the parent lipolytic enzyme has a structure comprising a catalytic triad consisting of an active Ser, an active Asp and an active His residue, and the amino acid to be altered is either located between the active His residue of the catalytic residue and the C-terminal, or belongs to a set E defined by the following steps:

i) aligning the structure of the lipolytic enzyme with Rhizomucor miehei lipase structure 4TGL comprising a catalytic triad and an inhibitor phosphorus atom (4TGL-inhP), so as to minimize the sum of squares of deviation between atoms of the catalytic triads of the two structures,

ii) defining a set A consisting of atoms of the lipolytic enzyme inside a sphere of radius 18 Å with center at 4TGL-inhP,

iii) forming a first plane defined by 4TGL-inhP, the Cα atom of the active Ser residue of the parent lipolytic enzyme, and the Cα atom of the active Asp residue of the parent lipolytic enzyme and defining a set B as a subset of set A consisting of atoms on the same side of the first plane as the Cα atom of the active His residue of the parent lipolytic enzyme,

iv) forming a second plane defined by 4TGL-inhP, the Cα atom of the active Ser residue of the parent lipolytic enzyme, and the Cα atom of the active His residue of the parent lipolytic enzyme and defining a set C as a subset of set A consisting of atoms on the opposite side of the second plane from the Cα atom of the active Asp residue of the parent lipolytic enzyme,

v) forming a set D consisting of atoms belonging to the union of sets B and C, and having a solvent accessibility of 15 or higher, and

vi) forming set E consisting of amino acid residues in the structure which comprise an atom belonging to set D or an atom belonging to the union of sets B and C and located less than 3.5 Å from an atom belonging to set D,

In a third aspect, the lipolytic enzyme has an active site comprising an active His residue, and the alteration is made in the amino acid sequence between the active His residue and the C-terminal.

In yet another aspect of the invention, the amino acid alteration is made among the 10 amino acid residues at the C-terminal.

In a further aspect, the parent lipolytic enzyme has a lid, and the alteration is made in the lid.

The invention also provides a DNA sequence encoding the variant, an expression vector comprising the DNA sequence, a transformed host cell harboring the DNA sequence or the expression vector, and to a method of producing the variant by cultivating the transformed host cell so as to produce the variant and recovering the variant from the resulting broth. Further, the invention provides uses of the variants.

The inventors have also found that a lipolytic enzyme which has lipase and phospholipase activity as well as activity on digalactosyl diglyceride is particularly effective for use in baking, and they designed a screening method for lipolytic enzymes by testing for these activities.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an alignment of lipase sequences as follows.

“rhimi” shows the Rhizomucor miehei lipase (SEQ ID NO:30)“rhidl” shows the Rhizomucor delemar lipase (SEQ ID NO:31)

“SP400” shows the Thermomyces (Humicola) lanuginosus lipase (SEQ ID NO:32)

“Pcl” shows the Penicillium camemberti lipase (SEQ ID NO:33)

“FoLnp11” shows the Fusarium oxysporum lipase (SEQ ID NO:34)

DETAILED DESCRIPTION OF THE INVENTION Altered Activity on Selected Ester Bond in Substrate

Compared to the parent lipolytic enzyme, the invention aims to alter the activity on at least one selected ester bond in at least one substrate, i.e. to increase a desired activity, decrease an undesired activity or to change the substrate specificity by decreasing the ratio of an undesired activity to a desired activity.

Thus, an enzyme with increased phospholipase activity may be useful, e.g., in baking or in purification of vegetable oil. It may be desired to increase the hydrolytic activity on digalactosyl-diglyceride (DGDG) for use in baking.

It may be desired to increase the lipase activity for any industrial use where lipases are used. For use in detergents or baking it may be desired to increase the activity on long-chain (C₁₆-C₂₀) triglycerides, and it may be desired to increase the specificity for long-chain fatty acids by decreasing the ratio of activity on short-chain or medium-chain (C₄-C₈) fatty acids to the activity on long-chain fatty acids.

For use in, or for use in flavor development in food products (such as cheese ripening) it may be desired to increase the lipase activity on short-chain or medium-chain (C₄-C₈) triglycerides.

For use as a phospholipase in purification of vegetable oil, it may be desired to decrease the ratio of lipase activity on long-chain (C₁₆-C₂₀) triglycerides to the phospholipase activity.

Parent Lipolytic Enzyme

The lipolytic enzyme to be used in the present invention is one that can hydrolyze ester bonds. Such enzymes include, for example, lipases, such as triacylglycerol lipase (EC 3.1.1.3), lipoprotein lipase (EC 3.1.1.34), monoglyceride lipase (EC 3.1.1.23), lysophospholipase, ferulic acid esterase and esterase (EC 3.1.1.1, EC 3.1.1.2). The numbers in parentheses are the systematic numbers assigned by the Enzyme Commission of the International Union of Biochemistry in accordance with the type of the enzymatic reactivity of the enzyme.

The parent lipolytic enzyme may be prokaryotic, particularly a bacterial enzyme, e.g. from Pseudomonas. Examples are Pseudomonas lipases, e.g. from P. cepacia (U.S. Pat. No. 5,290,694, pdb file 1OIL), P. glumae (N Frenken et al. (1992), Appl. Envir. Microbiol. 58 3787-3791, pdb files 1TAH and 1QGE), P. pseudoalcaligenes (EP 334 462) and Pseudomonas sp. strain SD 705 (FERM BP-4772) (WO 95/06720, EP 721 981, WO 96/27002, EP 812 910). The P. glumae lipase sequence is identical to the amino acid sequence of Chromobacterium viscosum (DE 3908131 A1). Other examples are bacterial cutinases, e.g. from Pseudomonas such as P. mendocina (U.S. Pat. No. 5,389,536) or P. putida (WO 88/09367).

Alternatively, the parent lipolytic enzyme may be eukaryotic, e.g. a fungal lipolytic enzyme such as lipolytic enzymes of the Humicola family and the Zygomycetes family and fungal cutinases.

Examples of fungal cutinases are the cutinases of Fusarium solani pisi (S. Longhi et al., Journal of Molecular Biology, 268 (4), 779-799 (1997)) and Humicola insolens (U.S. Pat. No. 5,827,719).

The Humicola family of lipolytic enzymes consists of the lipase from H. lanuginosa strain DSM 4109 and lipases having more than 50% homology with said lipase. The lipase from H. lanuginosa (synonym Thermomyces lanuginosus) is described in EP 258 068 and EP 305 216, and has the amino acid sequence shown in positions 1-269 of SEQ ID NO: 2 of U.S. Pat. No. 5,869,438.

The Humicola family also includes the following lipolytic enzymes: lipase from Penicillium camembertii (P25234), lipase/phospholipase from Fusarium oxysporum (EP 130064, WO 98/26057), lipase from F. heterosporum (R87979), lysophospholipase from Aspergillus foetidus (W33009), phospholipase A1 from A. oryzae (JP-A 10-155493), lipase from A. oryzae (D85895), lipase/ferulic acid esterase from A. niger (Y09330), lipase/ferulic acid esterase from A. tubingensis (Y09331), lipase from A. tubingensis (WO 98/45453), lysophospholipase from A. niger (WO 98/31790), lipase from F. solanii having an isoelectric point of 6.9 and an apparent molecular weight of 30 kDa (WO 96/18729).

The Zygomycetes family comprises lipases having at least 50% homology with the lipase of Rhizomucor miehei (P19515). This family also includes the lipases from Absidia reflexa, A. sporophora, A. corymbifera, A. blakesleeana, A. griseola (all described in WO 96/13578 and WO 97/27276) and Rhizopus oryzae (P21811). Numbers in parentheses indicate publication or accession to the EMBL, GenBank, GeneSeqp or Swiss-Prot databases.

It is of particular interest to derive a variant with phospholipase activity from a parent lipolytic enzyme having no or very little phospholipase activity, e.g. corresponding to a ratio of phospholipase activity to lipase activity below 0.1 PHLU/LU or below 50 PHLU/mg.

Alteration Near Alcohol Binding Site

As already stated, the amino acid sequence of the parent lipolytic enzyme may be modified at a position which near the glycerol part of a substrate triglyceride. This region will be referred to as the “alcohol binding site” of the lipase; it is described in Brzozowski A M et al., Nature, 351: 491 (1991); Uppenberg et al., Biochemistry, 1995, 34, 16838-16851; A. Svendsen, Inform, 5(5), 619-623 (1994).

For the Rhizomucor miehei lipase, the extent of the alcohol binding site can be found from the PDB file “5tgl.pdb” available in Structural Classification of Proteins (SCOP) on the Internet, at rcsb.org/pdb/, showing the complex with the inhibitor n-hexylphosphonate ethyl ester which mimics the substrate. It is described in Derewenda et al. (supra), Brzozowski et al. (supra) and Brady et al. (supra). The sn2 position of this model is the atom CE2.

The variant typically contains no more than 10 alterations in the alcohol binding site, e.g. 1, 2, 3, 4, 5 or 6 alterations.

The alteration may particularly be in that part of the alcohol binding site which comes within 20 positions (e.g. within 10 positions) of the C-terminal.

As already stated, the amino acid sequence of the parent lipolytic enzyme may be modified at a position which is within 10 Å (e.g. within 8 Å, particularly within 6 Å) of the C atom at the sn2 position of the glycerol part of a substrate triglyceride. The following amino acid positions lie within 10 Å of the sn2 position in the Rhizomucor miehei lipase: 25, 28, 80-84, 88, 143-146, 175, 203, 205, 254-255, 257-259, 264-267. The following are within 8 Å: 81-83, 144, 257-258, 265-267, and the following within 6 Å: 82, 144, 257, 266.

In the Humicola lanuginosa lipase, the following positions are within 10 Å of the sn2 position: 18, 21, 81-85, 89, 145-148, 172, 201, 203, 255-256, 258-260, 264-267. The following are within 8 Å: 82-84, 89, 146, 258-259, 265-267, and the following within 6 Å: 83, 146, 258, 266.

Alteration Near Catalytic Triad

As already stated, in one aspect the parent lipolytic enzyme has a structure comprising a catalytic triad consisting of an active Ser, an active Asp and an active His residue, and the amino acid to be altered belongs to a set defined by a certain procedure described above. The structure may be an open or a closed structure, and it may or may not include a substrate or an inhibitor.

The procedure is conveniently performed by use of software such as MSI's Insight II. It involves alignment with 4TGL, a crystal structure of the lipase from Rhizomucor miehei inhibited irreversibly by diethyl p-nitrophenyl phosphate. This is available in Structural Classification of Proteins (SCOP) on the Internet, at rcsb.org/pdb/, and is described in Derewenda et al. (supra). The Rhizomucor miehei lipase comprises a catalytic triad consisting of the amino acid residues S144, D203 and

H 257.

For the Humicola lanuginosa lipase, the structure 1tib may be used; it is available in Structural Classification of Proteins (SCOP) on the Internet. Using this structure, the set defined by the procedure includes the following positions: 10-23, 26, 40, 55-64, 80-87, 116-117, 119, 145-149, 151, 168, 170, 194, 196-201, 220-222, 224-227, and 254-269.

Alteration Between at C-Terminal Side of the Active His Residue

As stated above, one or more alterations may be made in the amino acid sequence between an active His residue and the terminal, specifically among the 12 amino acids at the C-terminal side of the active His.

The Humicola lanuginosa lipase has an active His at H258 and the C-terminal at L269, so this region includes positions 259-269. The P. cepacia lipase has an active H286 and the C-terminal at residue 297, so the region includes residues 287-297.

Alteration Near C-Terminal

As stated above, one or more alterations may be made within 10 amino acid positions from the C-terminal of the mature protein, or at positions corresponding to such positions in the H. lanuginosa lipase, i.e. positions 260-269 of the H. lanuginosa lipase. Corresponding positions may be found by alignment of the two sequences as described later in this specification.

The lipolytic enzyme variant may be truncated by deleting amino acid residues corresponding to the first 1, 2, 3, 4, 5 or 6 positions at the C-terminal. A truncated variant may have improved thermostability.

Alternatively, the variant may carry a peptide extension at the C-terminal and/or the N-terminal. The C-terminal extension may consist of 1-10 amino acid residues, e.g. A, P, AG, DG, PG, AGG, PVGF (SEQ ID NO. 15), AGRF (SEQ ID NO. 16), PRGF (SEQ ID NO. 17), AGGF (SEQ ID NO. 18) or AGGFS (SEQ ID NO. 19); or it may consist of 40-50 residues, e.g., consisting of the 48 C-terminal residues of the Fusarium oxysporum lipase AGGFSWRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS (SEQ ID NO. 20). The C-terminal extension may increase the phospholipase activity.

Some alterations in the region overlapping with the alcohol binding site are described below.

A specific alteration is a substitution at a position corresponding to G266 in the Humicola lanuginosa lipase, specifically with an amino acid of intermediate size, e.g. A, C, D, N, L, I, S, T, P or V. Such alteration alone has been found sufficient to increase the phospholipase activity.

Other specific alterations are such that alter the tertiary structure, e.g. by introducing bulky side chains or by disrupting the bond angles, e.g. by introducing Pro. Such alterations may be made at positions corresponding to positions G263, L264, I265, T267 or L269 in the Humicola lanuginosa lipase. Some specific substitutions are G263A,E,Q,R; L264A,C,P,Q; I265L,N,T; T267A,Q or L269N.

Alteration in Lid

As stated above, the amino acid sequence of the parent lipolytic enzyme may be modified in the lid region of the parent lipolytic enzyme. This region is described in Brady et al., Nature 343, 1990, pp. 767-770 and in Brzozowski A M et al., Nature, 351: 491 (1991). In the H. lanuginosa lipase, the lid is located at positions 80-100, and the modification may particularly be made at positions 82-98, e.g. 91-98.

The variant typically contains no more than 5 alterations in the lid region; it may contain 0, 1, 2 or 3 alterations. A specific alteration is a substitution of an amino acid corresponding to G91, L93, N94, D96, K98, L97 and/or E99 in the Humicola lanuginosa lipase with a neutral or positively charged amino acid, e.g. a substitution corresponding to G91A,T, L93K, N94D, D96S,W,G, L97Q, K98D,F,E and/or E99K,D.

Specifically, a variant with an alteration in the lid region also contains one or more alterations near the catalytic triad, near the substrate binding site or near the C-terminal.

Lipolytic Enzyme Variants

The lipolytic enzyme variant of the invention comprises one or more alterations of an amino acid residue in any of the regions described above. Each alteration may be a deletion or a substitution of the amino acid residue, or it may be an insertion before or after the amino acid residue. If the amino acid residue is at the C-terminal, the insertion may be a C-terminal extension. An insertion typically consists of 1-5 amino acid residues, e.g. 1-2, and a C-terminal extension may consist of 1-50 or 2-10 amino acid residues.

The total number of alterations in the above regions is typically not more than 20, e.g. not more than 10 or not more than 5, and there may be as little as 1 or 2 alterations in the above regions.

In addition, the lipolytic enzyme variant of the invention may optionally include other modifications of the parent enzyme, typically not more than 10, e.g. not more than 5 such modifications.

The variant generally has a homology with the parent lipolytic enzyme of at least 80%, e.g. at least 85%, typically at least 90% or at least 95%.

The variant of the invention may further comprise a peptide extension at the N-terminal, e.g. consisting of 1-15 (particularly 4-10) amino acid residues, and specifically comprising 1, 2 or 3 positively charged amino acids. Some specific N-terminal peptide extensions are AS, SPIRR (SEQ ID NO. 21), E1RP, E1SPIRPRP (SEQ ID NO. 22), E1SPPRRP (SEQ ID NO. 23) and E1SPIRPRP (SEQ ID NO. 22). Further, any peptide extension described in WO 97/04079 and WO 97/07202 may be used.

Specific Variants

To prepare variants of a lipolytic enzyme of the Humicola family, the amino acid alterations may specifically be made at positions corresponding to 20-25, 56-64, 81-85 or 255-269 in the Humicola lanuginosa lipase. Thus, the alteration may be a substitution, deletion or insertion at a position corresponding to A20, Y21, G23, K24, N25, V63, R81, G82, R84, A257, W260, Y261, F262 or G266 (e.g. excluding G23C, K24C, R81C), a substitution of an amino acid corresponding to C268 or L269.

Some specific alterations are substitutions corresponding to the following in H. lanuginosa lipase: Y21V/I/L/A/G/M/W/P/F/N/Q/S/T, V60V/I/L/A/G/M/W/P/F/N/Q/S/T, G61V/I/L/A/G/M/W/P/F/N/Q/S/T, D62E/A/V, S83T, R84K/L/W, P256A, G263E,Q,R,F, L264A,C,P,F,G,I, I265L,N,F G266D/E or T267A,Q,P,S,E, or an insertion corresponding to T267GS or T267GL.

To alter the activity towards short-chain (C₄-C₈) fatty acids in triglycerides, alterations may be made at positions corresponding to Y21, E56, D57, V60, G61, D62, R81, S83, R84, L259, Y261 or G266, e.g. a substitution corresponding to Y21V/I, V60G, D62E/A/V, S83T, R84K/L/W or G266D/E.

To increase the activity for DGDG, alterations may be made at positions corresponding to Y21, G23, N26, D57, D62, R81, S83, R84, S85, G266, T267 or L269; e.g., two or more such alterations may be made, e.g. together with one or more alterations in the lid region. To increase the phospholipase activity, alterations may be made at positions corresponding to R81, R84, S85, or 263-267, e.g. G266 or T267.

To prepare variants of a Pseudomonas lipase, amino acid modifications may be made at positions corresponding to 12-13, 16-34, 45-52, 59-66, 68, 86-87, 107-109, 111, 143-153, 155, 157-158, 207-212, 228, 230, 242-249, 264, 279-280, 282-297, 301-302, 304-305, 307-308 in the P. cepacia lipase, particularly L17/L17, T18/A18, Y29/Y29, L287/L286, E289/E288, I290/I289, Q292/Q291 or L293/L292 in the P. cepacia/P. glumae lipase.

Specific variants of the H. lanuginosa lipase are disclosed in the examples. Corresponding alterations may be made in other parent lipolytic enzymes. Further variants may be derived from these by omitting amino acid modifications at positions 1, 106, 186, 225, 232, 237, 239 or 274. Variants with 274S may optionally have a further C-terminal extension of

(SEQ ID NO. 24) WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS (corresponding to the C-terminal of the F. oxysporum lipase) in full or truncated form. Nomenclature for Amino Acid Alterations

The nomenclature used herein for defining mutations is basically as described in WO 92/05249. Thus, G91A indicates substitution of G in position 91 with A. T267A,Q indicates substitution of T at position 267 with A or Q. E1E,D,A indicates that E1 is unchanged or is substituted with D or A.

T267stop indicates a stop codon, i.e. deletion of T267 and all following amino acids (i.e. C268 and L269). 270P, 271V indicates a C-terminal extension of PV (i.e. at new positions 270 and 271). −G266 indicates deletion of G at position 266. Parentheses indicate that the alteration is optional, or in examples that the alteration is uncertain. SPIRR (SEQ ID NO. 21) indicates an N-terminal extension. D266 may refer to the position or to substitution with any amino acid (except D).

E1SPPCGRRP (SEQ ID NO. 25) or SPPCGRRP(-E) (SEQ ID NO. 25) indicates a substitution of E1 with SPPCGRRP (SEQ ID NO. 25), i.e. a peptide addition at the N-terminal. T267GS indicates a substitution of T267 with GS, or in other words the substitution T267G and an insertion of S between G267 and C268.

Homology and Alignment

For purposes of the present invention, the degree of homology may be suitably determined by means of computer programs known in the art, such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-45), using GAP with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1.

In the present invention, corresponding (or homologous) positions in the lipase sequences of Rhizomucor miehei (rhimi), Rhizopus delemar (rhidl), Thermomyces lanuginosa (former; Humicola lanuginosa) (SP400), Penicillium camembertii (Pcl) and Fusarium oxysporum (FoLnp11), are defined by the alignment shown in FIG. 1.

To find the homologous positions in lipase sequences not shown in the alignment, the sequence of interest is aligned to the sequences shown in FIG. 1. The new sequence is aligned to the present alignment in FIG. 1 by using the GAP alignment to the most homologous sequence found by the GAP program. GAP is provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-45). The following settings are used for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1.

Variants with Phospholipase Activity

As described above, the variant of the invention may have a higher phospholipase activity than the parent lipolytic enzyme. By the monolayer method described later in this specification, the variant may have a phospholipase activity of at least 0.1 nmol/min at pH 5.

By the PHLU method described later in this specification, the variant may have a phospholipase activity of at least 100 PHLU/mg (mg of pure enzyme protein), particularly at least 500 PHLU/mg. The variant has a ratio of phospholipase activity to lipase activity (both measured at pH 7) of at least 0.1 PHLU/LU, e.g. at least 0.5, particularly at least 2.

The variants of the invention may have the ability to hydrolyze intact phospholipid, as demonstrated by the PHLU method. They may have A₁ and/or A₂ activity, so they may be able to hydrolyze one or both fatty acyl groups in the phospholipid.

pH Optimum

Many variants of the Humicola lanuginosa lipase have an alkaline pH optimum for lipase activity and an acid pH optimum for phospholipase activity (e.g. pH 9-10 for lipase and pH 4-6 for phospholipase). Such variants can be used at acid pH (e.g. in oil degumming, described later), as phospholipases with very low concomitant lipase activity.

However, some variants of the Humicola lanuginosa lipase which include the substitution G266D,E have pH optima for both lipase and phospholipase activities around pH 5-6. Such variants may be used at acid pH when both lipase and phospholipase activities are desired, e.g. in baking.

Thermostability

The thermostability of the variant can conveniently be evaluated by means of Differential Scanning Calorimetry (DSC). Depending on exact mutations, the variants of the invention generally have similar or slightly lower thermostability than the parent lipolytic enzyme.

The temperature at the top of the denaturation peak (T_(d)) of the lipase from Humicola lanuginosa when heated at 90 deg/hr at pH 5 is just above 70° C. (=T_(d)). T_(d) for the variants of the invention is generally 5-10 degrees lower

Use of Variant

Depending on the substrate specificity, variants of the invention can be used, e.g., in filtration improvement, vegetable oil treatment, baking, detergents, or preparation of lysophospholipid.

Improvement of Filtration

A variant with lysophospholipase activity can be used to improve the filterability of an aqueous solution or slurry of carbohydrate origin by treating it with the variant. This is particularly applicable to a solution or slurry containing a starch hydrolysate, especially a wheat starch hydrolysate since this tends to be difficult to filter and to give cloudy filtrates. The treatment can be done in analogy with EP 219,269 (CPC International).

Vegetable Oil Treatment

A variant with phospholipase activity can be used in a process for reducing the content of phospholipid in an edible oil, comprising treating the oil with the variant so as to hydrolyze a major part of the phospholipid, and separating an aqueous phase containing the hydrolyzed phospholipid from the oil. This process is applicable to the purification of any edible oil which contains phospholipid, e.g. vegetable oil such as soy bean oil, rape seed oil and sunflower oil. The treatment may be carried out at acid pH, e.g. pH 3-5. Advantageously, a variant can be selected so as to have a high phospholipase activity and a low lipase activity at low pH, due to different pH optima of the two activities.

The process for oil treatment can be conducted according to principles known in the art, e.g. in analogy with U.S. Pat. No. 5,264,367 (Metallgesellschaft, Röhm); K. Dahlke & H. Buchold, INFORM, 6 (12), 1284-91 (1995); H. Buchold, Fat Sci. Technol., 95 (8), 300-304 (1993); JP-A 2-153997 (Showa Sangyo); or EP 654,527 (Metallgesellschaft, Röhm).

Miscellaneous Uses of Phospholipase

A variant with phospholipase activity can be used to prepare lysophospholipid (e.g. lyso-lecithin) by treating the corresponding phospholipid with the variant, e.g. as described in EP 870840, JP-A 10-42884, JP-A 4-135456 or JP-A 2-49593. The variant can also be used to make mayonnaise, e.g. as described in EP 628256, EP 398666 or EP 319064.

A variant with phospholipase activity may also be used in the processing of dairy and other food products, e.g. as described in EP 567,662 (Nestlé), EP 426,211 (Unilever), EP 166,284 (Nestlé), JP-A 57-189638 (Yakult) or U.S. Pat. No. 4,119,564 (Unilever).

The variant may be used leather treatment, as described in JP-A 7-177884 (Kao).

Baking

A variant with phospholipase and/or DGDGase activity can be used in the preparation of dough, bread and cakes, e.g. to increase dough stability and dough handling properties, or to improve the elasticity of the bread or cake. Thus, the variant can be used in a process for making bread, comprising adding the variant to the ingredients of a dough, kneading the dough and baking the dough to make the bread. This can be done in analogy with U.S. Pat. No. 4,567,046 (Kyowa Hakko), JP-A 60-78529 (QP Corp.), JP-A 62-111629 (QP Corp.), JP-A 63-258528 (QP Corp.), EP 426211 (Unilever) or WO 99/53769 (Novo Nordisk).

It is particularly advantageous to use the variant together with an anti-staling endo-amylase and optionally also to add a phospholipid, to reduce-staling of the bread and particularly to improve softness of the bread in the first 24 hours after baking. The endo-amylase may be a maltogenic α-amylase (e.g. from Bacillus sp., such as Novamyl® from Novo Nordisk) or a fungal or bacterial α-amylase, e.g. from Aspergillus or Bacillus, particularly A. oryzae, B. licheniformis or B. amyloliquefaciens.

In baking, the variant may have a low activity on short-chain or medium-chain (C₄-C₈), e.g. corresponding to a SLU/LU ratio above 3. The use of such a variant may avoid or suppress the development of an undesired flavor due to the release of short-chain fatty acids. The variant may have activity on triglycerides and phospholipid as well as DGDG.

Cheese Flavor

A variant with activity towards short-chain fatty acyl groups may be used to release free fatty acids (FFA) for flavor development in food products, e.g. in cheese ripening, e.g. as described in M. Hanson, ZFL, 41 (10), 664-666 (1990)).

Lipolytic enzyme variants with increased release of short chain fatty compared to long chain fatty acids from milk fat are useful in cheese production, e.g. for flavor enhancement or shortening of the ripening times for ripened cheeses, like cheddar or parmesan. Another application for such lipolytic enzyme variants is for enzyme modified cheese (EMC) for use as flavoring for various food products including process cheese, dressing and snack.

Release of short chain fatty acids, like butyric acid, is essential for the development of cheese flavor, whereas release of long chain fatty acids, like oleic acid, give rise to off flavors. Lipolytic enzyme variants for cheese applications, including EMC, should have SLU/LU ratio of less than 0.5, e.g. less than 0.25, most preferable less than 0.1

Use in Detergent

The variant may be used as a detergent additive, e.g. at a concentration (expressed as pure enzyme protein) of 0.001-10 (e.g. 0.01-1) mg per gram of detergent or 0.001-100 (e.g. 0.01-10) mg per liter of wash liquor.

In detergents, the variant may have a high activity on long-chain triglycerides (C₁₆-C₂₀) to improve the removal of fatty soiling. The variant may have phospholipase activity. The variant may have low activity towards short-chain (C₄-C₈) fatty acids in triglycerides, e.g. corresponding to a SLU/LU ratio above 10. The use of such a variant may avoid or suppress the development of an undesired odor due to the release of short-chain fatty acids.

Variants having both lipase and phospholipase activity at alkaline pH may be used in detergents.

Detergent Composition

The detergent composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations. In a laundry detergent, the variant may be effective for the removal of fatty stains, for whiteness maintenance and for dingy cleanup. A laundry detergent composition may be formulated as described in WO 97/04079, WO 97/07202, WO 97/41212, PCT/DK WO 98/08939 and WO 97/43375.

The detergent composition of the invention may particularly be formulated for hand or machine dishwashing operations. e.g. as described in GB 2,247,025 (Unilever) or WO 99/01531 (Procter & Gamble). In a dishwashing composition, the variant may be effective for removal of greasy/oily stains, for prevention of the staining/discoloration of the dishware and plastic components of the dishwasher by highly colored components and the avoidance of lime soap deposits on the dishware.

The detergent composition of the invention may be in any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. A liquid detergent may be aqueous, typically containing up to 70% water and 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which may be non-ionic including semi-polar and/or anionic and/or cationic and/or zwitterionic. The surfactants are typically present at a level of from 0.1% to 60% by weight, e.g. 0.5-40%, such as 1-30%, typically 1.5-20%.

When included therein the detergent will usually contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.

When included therein the detergent will usually contain from about 0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonyl-phenol ethoxylate, alkylpolyglycoside, alkyldimethylamine-oxide, ethoxylated fatty acid monoethanol-amide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucamides”).

The invention also provides a detergent additive comprising the variant of the invention. The detergent additive as well as the detergent composition may comprise one or more other enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., a laccase, and/or a peroxidase.

In general the properties of the chosen enzyme(s) should be compatible with the selected detergent, (i.e. pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.

Proteases: Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. The protease may be a serine protease or a metallo protease, e.g. an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274.

Specific commercially available protease enzymes include ALCALASE®, SAVINASE®, PRIMASE®, DURALASE®, ESPERASE®, and KANNASE® (Novozymes A/S), MAXATASE®, MAXACAL®, MAXAPEM®, PROPERASE®, PURAFECT®, PURAFECT OxP®, FN2™, and FN3™ (Genencor International Inc.).

Cellulases: Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulases having colour care benefits. Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

Commercially available cellulases include CELLUZYME®, and CAREZYME®(Novozymes A/S), CLAZINASE®, and PURADAX HA® (Genencor International Inc.), and KAC-500(B)® (Kao Corporation).

Peroxidases/Oxidases: Suitable per-oxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g. from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme® (Novo Nordisk A/S).

The detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive of the invention, i.e. a separate additive or a combined additive, can be formulated e.g. as a granulate, a liquid, a slurry, etc. Specific detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonyl-phenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. Liquid enzyme preparations may, for in-stance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238,216.

The detergent may contain 0-65% of a detergent builder or complexing agent such as zeolite, diphosphate, tripho-sphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetri-aminepen-taacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples are carboxymethylcellulose, poly(vinyl-pyrrolidone), poly(ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid co-polymers.

The detergent may contain a bleaching system which may comprise a H2O2 source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine or nonanoyloxyben-zenesul-fonate. Alternatively, the bleaching system may comprise peroxyacids of e.g. the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in e.g. WO 92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.

It is at present contemplated that in the detergent compositions any enzyme, in particular the variant of the invention, may be added in an amount corresponding to 0.01-100 mg of enzyme protein per liter of wash liquor, e.g. 0.05-5 mg of enzyme protein per liter of wash liquor, in particular 0.1-1 mg of enzyme protein per liter of wash liquor.

The variant of the invention may additionally be incorporated in the detergent formulations disclosed in WO 97/07202 which is hereby incorporated as reference.

Methods for Preparing Enzyme Variants

The enzyme variant of the invention can be prepared by methods known in the art, e.g. as described in WO 97/04079 (Novo Nordisk). The following describes methods for the cloning of enzyme-encoding DNA sequences, followed by methods for generating mutations at specific sites within the enzyme-encoding sequence.

Cloning a DNA Sequence Encoding a Enzyme

The DNA sequence encoding a parent enzyme may be isolated from any cell or microorganism producing the enzyme in question, using various methods well known in the art. First, a genomic DNA and/or cDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the enzyme to be studied. Then, if the amino acid sequence of the enzyme is known, labeled oligonucleotide probes may be synthesized and used to identify enzyme-encoding clones from a genomic library prepared from the organism in question. Alternatively, a labeled oligonucleotide probe containing sequences homologous to another known enzyme gene could be used as a probe to identify enzyme-encoding clones, using hybridization and washing conditions of lower stringency.

Yet another method for identifying enzyme-encoding clones would involve inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming enzyme-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing a substrate for enzyme (i.e. maltose), thereby allowing clones expressing the enzyme to be identified.

Alternatively, the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method described S. L. Beaucage and M. H. Caruthers, (1981), Tetrahedron Letters 22, p. 1859-1869, or the method described by Matthes et al., (1984), EMBO J. 3, p. 801-805. In the phosphoroamidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate, the fragments corresponding to various parts of the entire DNA sequence), in accordance with standard techniques. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in U.S. Pat. No. 4,683,202 or R. K. Saiki et al., (1988), Science 239, 1988, pp. 487-491.

Site-Directed Mutagenesis

Once a enzyme-encoding DNA sequence has been isolated, and desirable sites for mutation identified, mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites. In a specific method, a single-stranded gap of DNA, the enzyme-encoding sequence, is created in a vector carrying the enzyme gene. Then the synthetic nucleotide, bearing the desired mutation, is annealed to a homologous portion of the single-stranded DNA. The remaining gap is then filled in with DNA polymerase I (Klenow fragment) and the construct is ligated using T4 ligase. A specific example of this method is described in Morinaga et al., (1984), Biotechnology 2, p. 646-639. U.S. Pat. No. 4,760,025 discloses the introduction of oligonucleotides encoding multiple mutations by performing minor alterations of the cassette. However, an even greater variety of mutations can be introduced at any one time by the Morinaga method, because a multitude of oligonucleotides, of various lengths, can be introduced.

Another method for introducing mutations into enzyme-encoding DNA sequences is described in Nelson and Long, (1989), Analytical Biochemistry 180, p. 147-151. It involves the 3-step generation of a PCR fragment containing the desired mutation introduced by using a chemically synthesized DNA strand as one of the primers in the PCR reactions. From the PCR-generated fragment, a DNA fragment carrying the mutation may be isolated by cleavage with restriction endonucleases and reinserted into an expression plasmid.

Further, Sierks. et al., (1989) “Site-directed mutagenesis at the active site Trp 120 of Aspergillus awamori glucoamylase. Protein Eng., 2, 621-625; Sierks et al., (1990), “Catalytic mechanism of fungal glucoamylase as defined by mutagenesis of Asp176, Glu179 and Glu180 in the enzyme from Aspergillus awamon”. Protein Eng. vol. 3, 193-198; also describes site-directed mutagenesis in an Aspergillus glucoamylase.

Expression of Enzyme Variants

According to the invention, a DNA sequence encoding the variant produced by methods described above, or by any alternative methods known in the art, can be expressed, in enzyme form, using an expression vector which typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.

Expression Vector

The recombinant expression vector carrying the DNA sequence encoding a enzyme variant of the invention may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. The vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated. Examples of suitable expression vectors include pMT838.

Promoter

In the vector, the DNA sequence should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of the DNA sequence encoding a enzyme variant of the invention, especially in a bacterial host, are the promoter of the lac operon of E. coli, the Streptomyces coelicolor agarase gene dagA promoters, the promoters of the Bacillus licheniformis α-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens α-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding A. oryzae TAKA amylase, the TPI (triose phosphate isomerase) promoter from S. cerevisiae (Alber et al. (1982), J. Mol. Appl. Genet 1, p. 419-434, Rhizomucor miehei aspartic proteinase, A. niger neutral α-amylase, A. niger acid stable α-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.

Expression Vector

The expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably connected to the DNA sequence encoding the α-amylase variant of the invention. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and plJ702.

The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the dal genes from B. subtilis or B. licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, e.g. as described in WO 91/17243.

The procedures used to ligate the DNA construct of the invention encoding a enzyme variant, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989).

Host Cells

The cell of the invention, either comprising a DNA construct or an expression vector of the invention as defined above, is advantageously used as a host cell in the recombinant production of a enzyme variant of the invention. The cell may be transformed with the DNA construct of the invention encoding the variant, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g. by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.

The cell of the invention may be a cell of a higher organism such as a mammal or an insect, but may be a microbial cell, e.g. a bacterial or a fungal (including yeast) cell.

Examples of suitable bacteria are Gram positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyces lividans or Streptomyces murinus, or gramnegative bacteria such as E. coli. The transformation of the bacteria may, for instance, be effected by protoplast transformation or by using competent cells in a manner known per se.

The yeast organism may favorably be selected from a species of Saccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae.

The host cell may also be a filamentous fungus e.g. a strain belonging to a species of Aspergillus, such as Aspergillus oryzae or Aspergillus niger, or a strain of Fusarium, such as a strain of Fusarium oxysporium, Fusarium graminearum (in the perfect state named Gribberella zeae, previously Sphaeria zeae, synonym with Gibberella roseum and Gibberella roseum f. sp. cerealis), or Fusarium sulphureum (in the prefect state named Gibberella puricaris, synonym with Fusarium trichothecioides, Fusarium bactridioides, Fusarium sambucium, Fusarium roseum, and Fusarium roseum var. graminearum), Fusarium cerealis (synonym with Fusarium crokkwellnse), or Fusarium venenatum.

In a specific embodiment of the invention the host cell is a protease deficient of protease minus strain.

This may for instance be the protease deficient strain Aspergillus oryzae JaL 125 having the alkaline protease gene named “alp” deleted. This strain is described in WO 97/35956 (Novo Nordisk).

Filamentous fungi cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. The use of Aspergillus as a host micro-organism is described in EP 238 023 (Novo Nordisk A/S), the contents of which are hereby incorporated by reference.

Method of Producing the Enzyme Variant of the Invention

The enzyme variant of the invention may be produced by a method comprising cultivating a host cell under conditions conducive to the production of the variant and recovering the variant from the cells and/or culture medium.

The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the enzyme variant of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. as described in catalogues of the American Type Culture Collection).

The enzyme variant secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.

Expression of Variant in Plants

The present invention also relates to a transgenic plant, plant part or plant cell which has been transformed with a DNA sequence encoding the variant of the invention so as to express and produce this enzyme in recoverable quantities. The enzyme may be recovered from the plant or plant part. Alternatively, the plant or plant part containing the recombinant enzyme may be used as such.

The transgenic plant can be dicotyledonous or monocotyledonous, for short a dicot or a monocot. Examples of monocot plants are grasses, such as meadow grass (blue grass, Poa), forage grass such as festuca, lolium, temperate grass, such as Agrostis, and cereals, e.g. wheat, oats, rye, barley, rice, sorghum and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous (family Brassicaceae), such as cauliflower, oil seed rape and the closely related model organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds, and tubers. In the present context, also specific plant tissues, such as chloroplast, apoplast, mitochondria, vacuole, peroxisomes and cytoplasm are considered to be a plant part. Furthermore, any plant cell, whatever the tissue origin, is considered to be a plant part.

Also included within the scope of the invention are the progeny of such plants, plant parts and plant cells.

The transgenic plant or plant cell expressing the variant of the invention may be constructed in accordance with methods known in the art. In short the plant or plant cell is constructed by incorporating one or more expression constructs encoding the variant of the invention into the plant host genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell.

Conveniently, the expression construct is a DNA construct which comprises a gene encoding the variant of the invention in operable association with appropriate regulatory sequences required for expression of the gene in the plant or plant part of choice. Furthermore, the expression construct may comprise a selectable marker useful for identifying host cells into which the expression construct has been integrated and DNA sequences necessary for introduction of the construct into the plant in question (the latter depends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminator sequences and optionally signal or transit sequences is determined, eg on the basis of when, where and how the enzyme is desired to be expressed. For instance, the expression of the gene encoding the variant of the invention may be constitutive or inducible, or may be developmental, stage or tissue specific, and the gene product may be targeted to a specific tissue or plant part such as seeds or leaves. Regulatory sequences are eg described by Tague et al, Plant, Phys., 86, 506, 1988.

For constitutive expression the 35S-CaMV promoter may be used (Franck et al., 1980. Cell 21: 285-294). Organ-specific promoters may eg be a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990. Annu. Rev. Genet. 24: 275-303), or from metabolic sink tissues such as meristems (Ito et al., 1994. Plant Mol. Biol. 24: 863-878), a seed specific promoter such as the glutelin, prolamin, globulin or albumin promoter from rice (Wu et al., Plant and Cell Physiology Vol. 39, No. 8 pp. 885-889 (1998)), a Vicia faba promoter from the legumin B4 and the unknown seed protein gene from Vicia faba described by Conrad U. et al, Journal of Plant Physiology Vol. 152, No. 6 pp. 708-711 (1998), a promoter from a seed oil body protein (Chen et al., Plant and cell physiology vol. 39, No. 9 pp. 935-941 (1998), the storage protein napA promoter from Brassica napus, or any other seed specific promoter known in the art, eg as described in WO 91/14772. Furthermore, the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et al., Plant Physiology Vol. 102, No. 3 pp. 991-1000 (1993), the chlorella virus adenine methyltransferase gene promoter (Mitra, A. and Higgins, D W, Plant Molecular Biology Vol. 26, No. 1 pp. 85-93 (1994), or the aldP gene promoter from rice (Kagaya et al., Molecular and General Genetics Vol. 248, No. 6 pp. 668-674 (1995), or a wound inducible promoter such as the potato pin2 promoter (Xu et al, Plant Molecular Biology Vol. 22, No. 4 pp. 573-588 (1993).

A promoter enhancer element may be used to achieve higher expression of the enzyme in the plant. For instance, the promoter enhancer element may be an intron which is placed between the promoter and the nucleotide sequence encoding the enzyme. For instance, Xu et al. op cit disclose the use of the first intron of the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expression construct may be chosen from those available in the art.

The DNA construct is incorporated into the plant genome according to conventional techniques known in the art, including Agrobacterium-mediated transformation, virus-mediated transformation, micro injection, particle bombardment, biolistic transformation, and electroporation (Gasser et al, Science, 244, 1293; Potrykus, Bio/Techn. 8, 535, 1990; Shimamoto et al, Nature, 338, 274, 1989).

Presently, Agrobacterium tumefaciens mediated gene transfer is the method of choice for generating transgenic dicots (for review Hooykas & Schilperoort, 1992. Plant Mol. Biol. 19: 15-38), however it can also be used for transforming monocots, although other transformation methods are generally used for these plants. Presently, the method of choice for generating transgenic monocots is particle bombardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli or developing embryos (Christou, 1992. Plant J. 2: 275-281; Shimamoto, 1994. Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992. Bio/Technology 10: 667-674). An alternative method for transformation of monocots is based on protoplast transformation as described by Omirulleh S, et al., Plant Molecular biology Vol. 21, No. 3 pp. 415-428 (1993).

Following transformation, the transformants having incorporated the expression construct are selected and regenerated into whole plants according to methods well-known in the art.

Materials and Methods

Lipase Activity on Tributyrin (LU)

A substrate for lipase is prepared by emulsifying tributyrin (glycerin tributyrate) using gum Arabic as emulsifier. The hydrolysis of tributyrin at 30° C. at pH 7 is followed in a pH-stat titration experiment. One unit of lipase activity (1 LU) equals the amount of enzyme capable of releasing 1 μmol butyric acid/min at the standard conditions.

Lipase Activity on Triolein (SLU)

The lipolytic activity may be determined using olive oil as substrate.

In this SLU method, the lipase activity is measured at 30° C. and pH 9 with a stabilized olive oil emulsion (Sigma catalog No. 800-1) as the substrate, in a 5 mM Tris buffer containing 40 mM NaCl and 5 mM calcium chloride. 2.5 ml of the substrate is mixed with 12.5 ml buffer, the pH is adjusted to 9, 0.5 ml of diluted lipase sample is added, and the amount of oleic acid formed is followed by titration with a pH stat.

One SLU is the amount of lipase which liberates 1 μmole of titratable oleic acid per minute under these conditions.

Phospholipase Activity

The following assay methods were used for qualitative or quantitative determination of phospholipase activity.

Phospholipase Activity (PHLU)

Phospholipase activity (PHLU) is measured as the release of free fatty acids from lecithin. 50 μl 4% L-alpha-phosphatidylcholine (plant lecithin from Avanti), 4% Triton X-100, 5 mM CaCl₂ in 50 mM HEPES, pH 7 is added 50 μl enzyme solution diluted to an appropriate concentration in 50 mM HEPES, pH 7. The samples are incubated for 10 min at 30° C. and the reaction stopped at 95° C. for 5 min prior to centrifugation (5 min at 7000 rpm). Free fatty acids are determined using the NEFA C kit from Wako Chemicals GmbH; 25 μl reaction mixture is added 250 μl Reagent A and incubated 10 min at 37° C. Then 500 μl Reagent B is added and the sample is incubated again, 10 min at 37° C. The absorption at 550 nm is measured using an HP 8452A diode array spectrophotometer. Samples are run in at least in duplicates. Substrate and enzyme blinds (preheated enzyme samples (10 min at 95° C.)+substrate) are included. Oleic acid is used as a fatty acid standard. 1 PHLU equals the amount of enzyme capable of releasing 1 μmol of free fatty acid/min at these conditions.

Phospholipase Activity (LEU)

Lecithin is hydrolyzed under constant pH and temperature, and the phospholipase activity is determined as the rate of titrant (0.1N NaOH) consumption during neutralization of the liberated fatty acid.

The substrate is soy lecithin (L- -Phosphotidyl-Choline), and the conditions are pH 8.00, 40.0° C., reaction time 2 min. The unit is defined relative to a standard.

Phospholipase Monolayer Assay

On a thoroughly purified surface of a buffer solution (either 10 mM Glycin, pH 9.0 or 10 mM NaOAc, pH 5.0; 1 mM CaCl2, 25° C.) a monolayer of Di-Decanoyl-Phosphatidyl Choline (DDPC) is spread from a chloroform solution. After relaxation of the monolayer (evaporation of chlorofom) the surface pressure is adjusted to 15 mN/m, corresponding to a mean molecular area of DDPC of approx. 63 Å²/molec. A solution containing approximately 60 μg (micro gram) enzyme is injected through the monolayer into the subphase of the re-action compartment (cylinder with surface area 2230 mm2 and reaction volume 56570 mm3) in the “zero-order trough”. Enzymatic activity is manifested through the speed of a mobile barrier compressing the monolayer in order to maintain constant surface pressure as insoluble substrate molecules are hydrolyzed into more water soluble reaction products. Having verified that the aqueous solubility of the reaction products (capric acid and MDPC) are considerably higher than for DDPC the number of DDPC-molecules hydrolyzed per minute by the enzyme is estimated from the mean molecular area (MMA) of DDPC. The results are calculated on basis of average barrier speed over the first 5 minutes of hydrolysis.

The result is considered positive for phospholipase if the barrier moves at more than 2 mm/min.

Plate Assay 1

A) 50 ml 2% agarose in purified water is melted/stirred for 5 minutes and cooled to 60-63° C.

B) 50 ml 2% plant L-alpha-Phosphatidylcholine 95% in 0.2M NaOAc, 10 mM CaCl₂, pH 5.5 at 60° C. in 30 min. is blended in 15 sec. with ultrathorax.

Equal volumes of 2% agarose and 2% Lecithin (A and B) are mixed, and an equal volume of 1% Triton X-100 is added to this mixture. 250 μl 4 mg/ml crystal violet in purified water is added as indicator. The mixture is poured into appropriate petri dishes (e.g. 30 ml in 14 cm Ø dish), and appropriate holes are made in the agar (3-5 mm) for application of enzyme solution.

The enzyme sample is diluted to a concentration corresponding to OD₂₈₀=0.5 and 10 microliter is applied into holes in the agarose/lecithin-matrix. Plates are incubated at 30° C. and reaction zones in the plates are identified after approx. 4-5 hours and/or after approx. 20 hours incubation. The Humicola lanuginosa lipase is used as a control, and the presence of a larger clearing zone than the control is taken as a positive result for phospholipase activity.

In a variation of this assay, the addition of Triton X-100 is omitted.

Plate Assay 2

10 g agarose is melted in 550 ml H2O by boiling in a microwave oven. After cooling to 60-70° C. the following ingredients are added:

250 ml of a 0.4 M Citrate buffer (pH 4.5 or pH 7.1)

200 ml 3% lecithin (from Avanti) in 2% Triton-X 100

2 ml 2% crystal violet

30 ml of the mixture is poured into 14 cm Ø petri dishes.

The plates are incubated after application of enzyme samples, and the results are interpreted as for Plate assay 1.

Digalactosyl Diglyceride Hydrolyzing (DGDGase) Activity

Monolayer Assay 1

On a thoroughly purified surface of a buffer solution (10 mM NaOAc, pH 5.5; 1 mM CaCl2, 25° C.; 10 mM beta-cyclodextrin (Sigma C-4767)) a monolayer of DGDG (Sigma (D4651)) is spread from a chloroform solution. After relaxation of the monolayer (evaporation of chlorofom) the surface pressure is adjusted to 15 mN/m. A solution containing approximately 60 μg (micro gram) enzyme is injected through the monolayer into the subphase of the re-action compartment (cylinder with surface area 2230 mm² and reaction volume 56570 mm³) in the “zero-order trough”. Enzymatic activity is manifested through increased speed of a mobile barrier compressing the monolayer in order to maintain constant surface pressure as insoluble substrate molecules are hydrolyzed into more water soluble reaction products (in presence of beta cyclodextrin).

The result is considered positive for DGDGase if the barrier moves at more than 1 mm/min.

Monolayer 2

On a thoroughly purified surface of a buffer solution (approx. 75 ml, 10 mM NaOAc, pH 5.5; 1 mM CaCl2, 25° C.; 10 mM beta-cyclodextrin (Sigma C-4767)) a monolayer of DGDG (Sigma (D4651)) is spread from a chloroform solution to a surface pressure of about 30 mN/m. After relaxation of the monolayer (evaporation of chlorofom) a solution containing approximately 30 μg (micro gram) purified enzyme is injected through the monolayer into the 75 ml subphase while surface pressure is measured continuously. Enzymatic activity is manifested through increased rate of decrease in surface pressure as DGDG is hydrolyzed into water soluble reaction products (in presence of beta cyclodextrin).

The result is considered positive for DGDGase if maximal drop in surface pressure (dπ/dt) after addition of enzyme exceeds −0.5 mN/min. A number of variants of Lipolase were tested and found to have DGDGase activity, whereas the parent enzyme (Lipolase) only had very limited activity (dπ/dt>−0.5 mN/min.).

Yeast Strain

Saccharomyces cerevisiae YNG318: MATa leu2-D2 ura3-52 his4-539 pep4-D1[cir+], described in WO 97/04079 and WO 97/07205.

Transformation of Yeast Strain

The DNA fragments and the opened vectors are mixed and transformed into the yeast Saccharomyces cerevisiae YNG318 by standard methods.

Vector for Yeast Transformation

pJSO026 (S. cerevisiae expression plasmid) is described in WO 97/07205 and in J. S. Okkels, (1996) “A URA3-promoter deletion in a pYES vector increases the expression level of a fungal lipase in Saccharomyces cerevisiae. Recombinant DNA Biotechnology III: The Integration of Biological and Engineering Sciences, vol. 782 of the Annals of the New York Academy of Sciences). It is derived from pYES 2.0 by replacing the inducible GAL1-promoter of pYES 2.0 with the constitutively expressed TPI (triose phosphate isomerase)-promoter from Saccharomyces cerevisiae (Albert and Karwasaki, (1982), J. Mol. Appl. Genet., 1, 419-434), and deleting a part of the URA3 promoter.

Site-Directed Mutagenesis

For the construction of variants of a H. lanuginosa lipolytic enzyme the commercial kit, Chameleon double-stranded, site-directed mutagenesis kit can be used according to the manufacturer's instructions.

The gene encoding the lipolytic enzyme in question is inserted into the plasmid pHD414. In accordance with the manufacturer's instructions the ScaI site of the Ampicillin gene of pHD414 is changed to a MluI site by use of the following primer:

Primer 3: AGAAATCGGGTATCCTTTCAG. (SEQ ID NO. 27)

The pHD414 vector comprising the lipolytic gene in question is then used as a template for DNA polymerase and oligos 7258 and 7770.

7258: (SEQ ID NO. 28) 5′p gaa tga ctt ggt tga cgc gtc acc agt cac 3′

(Thus changing the ScaI site found in the ampicillin resistance gene and used for cutting to a MluI site).

Primer no. 7770 was used as the selection primer.

7770: 5′p tct agc cca gaa tac tgg atc aaa tc 3′ (SEQ ID NO. 29) (Changes the ScaI site found in the H. lanuginosa lipase gene without changing the amino acid sequence).

The desired mutation (e.g. in the N-terminal of the lipolytic gene or the introduction of a cystein residue) is introduced into the lipolytic gene in question by addition of an appropriate oligos comprising the desired mutation.

PCR reactions are performed according to the manufacturer's recommendations.

Screening Method

The yeast libraries are spread on cellulose filters on SC-ura agar plates and incubated for 3-4 days at 30° C.

The filters are then transferred to the lecithin plates and incubated at 37° C. for 2-6 hours. Yeast cells harboring active phospholipases will develop white clearing zones around the colonies. The positive variants can then be further purified and tested.

Media SC-ura medium Yeast Nitrogen (without amino aicds) 7.5 g Succinic acid 11.3 g NaOH 6.8 g Casaminoacid (without vitamins) 5.6 g Tryptophan 0.1 g Agar, Merck 20 g Distilled water ad 1000 ml Autoclaved for 20 minutes at 121° C.

From a sterile stock solution of 5% Threonine 4 ml is added to a volume of 900 ml together with 100 ml of a sterile 20% glucose.

EXAMPLES Example 1 Construction of Variants with the Backbone from Humicola lanuginosa Lipase and C-Terminal from Fusarium oxysporum Phospholipase by PCR Reaction

The following variants were used as templates for the backbone from the Humicola lanuginosa lipase: E1A+G91A+D96W+E99K+Q249R and SPIRR+G91A+D96W+E99K+Q249R. The parent lipase was used for generating a fragment in the C-terminal without Q249R. The template for the C-terminal phospholipase was the Fusarium oxysporum phospholipase, cloned in the same vector as the variants of Humicola lanuginosa lipase.

PCR reaction 1: 4244 (SEQ ID NO: 1) as 5′ primer and H7 (SEQ ID NO: 6) as 3′ primer and one of the two templates mentioned above.

PCR reaction 2: FOL14 (SEQ ID NO: 3) as 5′ primer and FOL15 (SEQ ID NO: 4) as 3′ primer and Humicola lanuginosa lipase as template (no mutation in pos 249)

PCR reaction 3: FOL16 (SEQ ID NO: 5) as 5′ primer and AP (SEQ ID NO: 2) as 3′ primer and F.o. phospholipase as template

A PCR reaction 4 was made to create the connection between the Humicola lanuginosa lipase variant and the C-terminal from the phospholipase by using FOL14 (SEQ ID NO: 3) as 5′ primer and AP (SEQ ID NO: 2) as 3′ primer and PCR reaction 2 and 3 as template.

The final PCR was made with 4244 (SEQ ID NO: 1) as 5′ primer and KBoj14 (SEQ ID NO: 7) as 3′ primer and PCR re-action 1 and 4 as template (by using Humicola lanuginosa lipase as template in reaction 2 a possibility to omit the mutation in position 249 was created).

The final PCR fragment was used in an in vivo recombination in yeast together with pJSO026 cut with the restriction enzymes. SmaI (or BamHI) and XbaI (to remove the coding region and at the same time create an overlap of about 75 bp in each end to make a recombination event possible). This final treatment was also used in the following examples.

Primer FOL14 (SEQ ID NO: 3) and primer 15/16 are mixed oligoes to give the possibility to bind both with Humicola lanuginosa lipase and phospholipase templates and at the same time give possibilities for introducing the amino acids from both templates in the different positions. For some of the positions new amino acids could be introduced as well.

Primer FOL14 (SEQ ID NO: 3)

Position 205 in the H. lanuginosa lipase: 75% R, 25% S

Primer FOL15 (SEQ ID NO: 4)/FOL16 (SEQ ID NO: 5)

Position 256 in the H. lanuginosa lipase: 50% P, 50% A

Position 260 in the H. lanuginosa lipase: 25% R, 12.5% Q, 12.5% H, 12.5% C, 12.5% Y, 12.5% W, 12.5% stop.

The sequences of the resulting variants were determined, and were found to correspond to Humicola lanuginosa lipase with the following alterations. Alterations in parentheses are uncertain.

E1A, G91A, D96W, E99K, P256A, W260H, G263Q, L264A, I265T, G266D, T267A, L269N, 270A, 271G, 272G, 273F, (274S) E1A, G91A, D96W, E99K, E239C, Q249R, P256A, G263Q, L264A, I265T, G266D, T267A, L269N, 270A, 271G, 272G273F, (274S) E1A, G91A, D96W, E99K, N248T, Q249R, W260Q, G263Q, L264A, I265T, G266D, T267A, L269N, 270A, 271G, 272G, 273F, (274S) SPIRR (SEQ ID NO. 21), G91A, D96W, E99K, W260C, G263Q, L264A, I265T, G266D, T267A, L269N, 270A, 271G, 272, G273F, (274S) SPIRR (SEQ ID NO. 21), G91A, D96W, E99K, G263Q, L264A, I265T, G266D, T267A, L269N, 270A, 271G, 2720, 273F, (274S) E1A, G91A, D96W, E99K, G263Q, L264A, I265T, G266D, T267A, L269N, 270A, 271G, 272G, 273F, (274S)

Example 2 Production of Truncated Sequences

Variants were made with stop after amino acid 269, 270, 271, 272, (273 and 274)

The following PCR reactions were made with the following template: E1A, G91A, D96W, E99K, P256A, W260H, G263Q, L264A, 1265T, G266D, T267A, L269N, 270A, 271G, 272G, 273F, (274S).

Reaction 1: 5′ primer 4244 (SEQ ID NO: 1) and 3′ primer KBoj36 (stop after 269)

Reaction 2: 5′ primer 4244 (SEQ ID NO: 1) and 3′ primer KBoj37 (stop after 270)

Reaction 3: 5′ primer 4244 (SEQ ID NO: 1) and 3′ primer KBoj38 (stop after 271)

Reaction 4: 5′ primer 4244 (SEQ ID NO: 1) and 3′ primer KBoj39 (stop after 272)

The sequences of the resulting variants were determined, and were found to correspond to Humicola lanuginosa lipase with the following alterations:

E1A, G91A, D96W, E99K, P256A, W260H, G263Q, L264A, I265T, G266D, T267A, L269N E1A, G91A, D96W, E99K, P256A, W260H, G263Q, L264A, I265T, G266D, T267A, L269N, 270A E1A, G91A, D96W, E99K, P256A, W260H, G263Q, L264A, I265T, G266D, T267A, L269N, 270A, 271G E1A, G91A, D96W, E99K, P256A, W260H, G263Q, L264A, I265T, G266D, T267A, L269N, 270A, 271G, 272G

Example 3 Removal of Mutations in the Lid Region

G91A or E99K can be removed without loosing the phospholipase activity. The sequences of the resulting variants were determined, and were found to correspond to Humicola lanuginosa lipase with the following alterations:

E1A, G91A, D96W, P256A, W260H, G263Q, L264A, I265T, G266D, T267A, L269N, 270A, 271G, 272G, 273F, (274S) SPIRR (SEQ ID NO. 21), D96W, E99K, G263Q, L264A, I265T, G266D, T267A, L269N, 270A, 271G, 272G, 273F, (274S) SPIRR (SEQ ID NO. 21), G91A, D96W, G263Q, L264A, I265T, G266D, T267A, L269N, 270A, 271G, 272G, 273F, (274S) E1A, G91A, D96W, P256A, W260H, G263Q, L264A, I265T, G266D, T267A, L269N, 270A, 271G, 272G, 273F, (274S)

Example 4 Doping in the C-Terminal Region of Humicola lanuginosa Lipase to Introduce Phospholipase Activity

Three different libraries were constructed with possibilities for mutations in position 256 and position 263-269. At the same time possibilities for extension of the C-terminal with either 1, 2, 3 or 4 amino acids were included.

Doping, the wt sequences are underlined:

256: P 94, A 3, T 3 263: G 87, E 4.8, A 3.8, R 3.6, Q 0.2, P 0.2 264: L 87, P 4.8, Q 3.8, V 3.6, A 0.2, E 0.2 265: I 85, T 5.6, L 2.2, S 1.6, N 1.5, F 1.4, R 0.4, K 0.4 A, P 0.1, G, D, C, H, Y 0.03, Q, E 0.01, stop 0.016 266: G 86, D 5.9, R 2, S 1.7, C 1.6, A 0.9, V 0.9, E 0.7, W 0.2, H, Y 0.1, I, L, T, F, P 0.02, Q, K 0.01, stop 0.014 267: T 86, A 6.6, S 1.9, R 0.9, N 0.9, I 0.9, K 0.9, M 0.9, P 0.9, P 0.9, G, V 0.14, D, E 0.07, L 0.03, C, Q, H, F, W, Y 0.01, stop 0.01 268: C 91, S 1.9, R 1.0, G 1.0, F 0.9, Y 0.9, L 0.04, A, N, D, H, I, P, T, V 0.01, stop 2.8 269: L 92, stop 8 (KBoj 32 (SEQ ID NO: 8) and KBoj33)/N 86, K 2.7, D 1.8, H 1.8, I 1.8, S 1.8, T 1.9, Y 1.8, R 0.1, Q, M, E 0.06, A, C, G, L, F, P, V 0.04, stop 0.06(KBoj34) 270: stop 100 (KBoj33)/A 44, P 44, S 1.9, T 1.8, R 1.5, L 1.5, G 1.4, V 1.4, D 0.7, Q 0.7, E 0.7, H 0.7, N, C, I, K, M, F, W, Y 0.03, stop 0.03 (KBoj 32 (SEQ ID NO: 8) and KBoj 34) 271: G 72, R 4.5, V 3.2, E 3.0, C 2.9, A 1.6, S 1.2, D 1.0, L 0.5, I, K, Y 0.15, Q, T 0.08, N, P 0.05, stop 9.2 272: G 72, R 4.5, V 3.2, E 3.0, C 2.9, A 1.6, S 1.2, D 1.0, L 0.5, I, K, Y 0.15, Q, T 0.08, N, P 0.05, stop 9.2 273: F 74, L 11, S 2.8, I 2.7, V 2.7, Y 2.5, C 2.5, A, R, T 0.1, N, D, H 0.08, Q, E, K 0.01, stop 0.5 274 STOP

Library A: PCR reaction with 4244 (SEQ ID NO: 1) as 5′ primer and KBoj 33 as 3′ primer and E1A+G91A+D96W+E99K+Q249R or E1A+G225R as template. Variants from this library will be without extension.

Library B: PCR reaction with 4244 (SEQ ID NO: 1) as 5′ primer and KBoj 32 (SEQ ID NO: 8) as 3′ primer and E1A+G91A+D96W+E99K+Q249R or E1A+G225R as template. Variants from this library will most probably contain a C-terminal extension but can contain stop codons before the extension.

Library C: PCR reaction with 4244 (SEQ ID NO: 1) as 5′ primer and KBoj 34 as 3′ primer and E1A+G91A+D96W+E99K+Q249R or E1A+G225R as template. Variants from this library will most probably contain mutations in position 269 and a C-terminal extension but can contain stop codons before the extension.

The following variants were obtained:

Library A:

E1A+G91A+D96W+E99K+Q249R+G266D

Library B:

E1A+G91A+D96W+E99K+(R232L)+Q249R+G266S+270A

E1A+G91A+D96W+E99K+Q249R+G266S+270D+271G

E1A+G91A+D96W+E99K+Q249R+L264G+I265G+G266F+T267stop

E1A+G91A+D96W+E99K+Q249R+G266A+270P+271G

E1A+G91A+D96W+E99K+Q249R+L264P+I265F+L269stop

Library C:

E1A+G91A+D96W+E99K+Q249R+G263E+G266D+L269N+270P+271V+272G+273F

E1A+G91A+D96W+E99K+Q249R+G263A+G266S+L269N+270A+271G+272R+273F

E1A+G91A+D96W+E99K+Q249R+L264P−G266+L269I+270P+271R+272G+273F

E1A+G91A+D96W+E99K+Q249R+G266D+L269S+270A+271G+272G+273F

E1A+D27G+G91A+D96W+E99K+Q249R+G266S+L269N+270A+271G+272G+273F

E1A+G91A+D96W+E99K+Q249R+G266D+L269N+270A

E1A+G91A+D96W+E99K+Q249R+L264P+L267Q+L269N

E1A+G91A+D96W+E99K+Q249R+G263R+I265L+L269N+270P

Example 5

For some of the above variants, the pH optimum for lipase and phospholipase was determined by using the LU and PHLU methods at various pH values. The results showed that the pH optimum phospholipase activity was in the range 4-6. The optimum for lipase activity varied from about pH 6 to about pH 10.

8 variants listed in Example 5 were analyzed for phospholipase activity by the mono layer assay described above at pH 5 and 9. The results showed that all the variants have phospholipase activity at pH 5 and 9, whereas the parent lipase (Humicola lanuginosa lipase) showed no activity at pH 5 or 9. Depending on the variant, the activity at pH 5 was higher or lower than at pH 9.

A prior-art variant of Humicola lanuginosa lipase was found to have no phospholipase activity at pH 5: SPIRR+N94K+F95L+D96H+N101S+F181L+D234Y+I252L+P256T+G263A+L264Q.

Example 5 Variants of Humicola Lipase with Phospholipase Activity

Variants of the parent lipase from Humicola lanuginosa were prepared and tested for phospholipase activity as described above. The following variants were found to have phospholipase activity, where as the parent had no phospholipase activity by the same method.

E1A, G91A, D96W, E99K, P256A, W260H, G263Q, L264A, I265T, G266D, T267A, L269N, 270A, 271G, 272G, 273F, (274S) SPIRR (SEQ ID NO. 21), G91A, D96W, E99K, G263Q, L264A, I265T, G266D, T267A, L269N, 270A, 271G, 272G, 273F, (274S) E1A, G91A, D96W, P256A, W260H, G263Q, L264A, I265T, G266D, T267A, L269N, 270A, 271G, 272G, 273F, (274S) E1A, G91A, D96W, E99K, P256A, W260H, G263Q, L264A, I265T, G266D, T267A, L269N E1A, G91A, D96W, E99K, Q249R, G266S, 270D, 271G E1A, G91A, D96W, E99K, Q249R, G266D E1A, G91A, D96W, E99K, Q249R, G266A, 270P, 271G G266D E1SPPCGRRP (SEQ ID NO. 25) + E99N + E239C + Q249R + G266D E1SPPCGRRP (SEQ ID NO. 25) + E239C + Q249R + G266D E1SPPCGRRP (SEQ ID NO. 25) + L93K + E99K + E239C + Q249R + G266D E1SPPCGRRP (SEQ ID NO. 25) + E99K + E239C + Q249R + G266D G266A G266W G266V G263Q + L264A + I265T + G266D + T267A G263F + L264A + G266S + T267E E1SPPCGRRP (SEQ ID NO. 25) + E239C + Q249R + G263Q + L264A + I265T + G266D + T267A G266S G266L G263A + G266A G263A + G266Y E1SPPCGRRP (SEQ ID NO. 25) + E239C + Q249R + G266A E1SPPCGRRP (SEQ ID NO. 25) + E239C + Q249R + G266S E1SPPCGRRP (SEQ ID NO. 25) + E239C + Q249R + G263F + L264A + G266S + T267E D62A + G266A D62A + G266S D96S + G266A D96S + G266S D96S + G266R D96S + G266W D96S + G266V E1SPPCGRRP (SEQ ID NO. 25) + G91A + D96W + E239C + Q249R + G266D E1SPPCGRRP (SEQ ID NO. 25) + G91A + D96W + E239C + Q249R + G266S E1SPPCGRRP (SEQ ID NO. 25) + G91A + D96W + E239C + Q249R + G263E + G266S + 270A E1SPPCGRRP (SEQ ID NO. 25) + G91A + D96W + E239C + Q249R + L264P + G266S E1SPPCGRRP (SEQ ID NO. 25) + G91A + D96W + E239C + Q249R + P256T + G266D E1SPPCGRRP (SEQ ID NO. 25) + G91A + D96W + E239C + Q249R + G266C + T267P + L269stop G263D + L264I + I265N + G266E + T267GS E219G + L264I + I265N + G266T + T267GL E1A + G91A + D96W + E99K + P256A + W260H + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F (+274S) E1A + G91A + D96W + E99K + E239C + Q249R + P256A + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F (+274S) E1A + G91A + D96W + E99K + N248T + Q249R + W260Q + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F (+274S) SPIRR (SEQ ID NO. 21) + G91A + D96W + E99K + W260C + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272 + G273F (+274S) SPIRR (SEQ ID NO. 21) + G91A + D96W + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F (+274S) E1A + G91A + D96W + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F (+274S) E1A + G91A + D96W + E99K + P256A + W260H + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F (+274S) SPIRR (SEQ ID NO. 21) + D96W + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F (+274S) SPIRR (SEQ ID NO. 21) + G91A + D96W + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F (+274S) E1A + G91A + D96W + E99K + P256A + W260H + G263Q + L264A + I265T + G266D + T267A + L269N E1A + G91A + D96W + E99K + Q249R + G263E + G266D + L269N + 270P + 271V + 272G + 273F E1A + G91A + D96W + E99K + Q249R + G263A + G266S + L269N + 270A + 271G + 272R + 273F E1A + G91A + D96W + E99K + Q249R + L264P + Δ266 + L269I + 270P + 271R + 272G + 273F E1A + G91A + D96W + E99K + Q249R + L264C + I265N + G266P + T267stop E1A + G91A + D96W + E99K (+R232L) + Q249R + G266S + 270A E1A + G91A + D96W + E99K + Q249R + G266S + 270D + 271G E1A + G91A + D96W + E99K + Q249R + L264F + Δ266 + 270A + 271G + 272G + 273F E1A + G91A + D96W + E99K + Q249R + L264G + I265G + G266F + T267stop E1A + G91A + D96W + E99K + Q249R + L264stop E1A + G91A + D96W + E99K + P256A + W260H + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G E1A + G91A + D96W + E99K + P256A + W260H + G263Q + L264A + I265T + G2660 + T267A + L269N + 270A + 271G + 272G E1A + G91A + D96W + E99K + Q249R + G266D E1A + G91A + D96W + E99K + Q249R + G266D E1A + G91A + D96W + E99K + Q249R + G266A + 270P + 271G E1A + G91A + D96W + E99K + Q249R + L264P + I265F + L269stop E1A + G91A + D96W + E99K + Q249R + G266D + L269S + 270A + 271G + 272G + 273F E1A + G91A + D96W + E99K + Q249R + G266D + L269N + 270A E1A + G91A + D96W + E99K + Q249R + G266S + L269N + 270A + 271G + 272G + 273F E1A + G91A + D96W + E99K + Q249R + L264P + L267Q + L269N E1A + G91A + D96W + E99K + Q249R + G263R + I265L + L269N + 270P E1A + D96W + E99K + P256A + W260H + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F (+274S) E1A + G225R + G266D E1A + G225R + G263A + I265V + G266S E1A + G225R + G263A + T267A E1SPPCGRRP (SEQ ID NO. 25) + D96S + E239C + Q249R + I252M + L264Q + G266D E1SPPCGRRP (SEQ ID NO. 25) + G91A + D96W + E2390 + Q249R + G266D E1SPPCGRRP (SEQ ID NO. 25) + D96S + E239C + Q249R + G266D E1SPPCGRRP (SEQ ID NO. 25) + D96S + E239C + Q249R + 02660 + L267A E1A + G91A + D96W + E99K + Q249R + G266A E1A + D96M + G106S + G225R + G266D E1A + D96Q + G106S + G225R + G266S E1A + D96F + G225R + G266S E1A + D96C + G225R + G266T E1A + D96H + G106S + G225R + G266S SPIRR (SEQ ID NO. 21) + D96S + G266D SPIRR (SEQ ID NO. 21) + D96R + G106S + G266D SPIRR (SEQ ID NO. 21) + D961 + G106S + G266S SPIRR (SEQ ID NO. 21) + D96W + K237R + G266S SPIRR (SEQ ID NO. 21) + G266A SPIRR (SEQ ID NO. 21) + D96S + G106S + G225R + G266D SPIRR (SEQ ID NO. 21) + D96Q + G106S + G225R + G266A SPIRR (SEQ ID NO. 21) + D96Y + G106S + G225R + G266N SPIRR (SEQ ID NO. 21) + D96C + G106S + G225R + G266T SPIRR (SEQ ID NO. 21) + D96H + T1861 + G225R + G2665 E1SPPRRP (SEQ ID NO. 23) + G91A + D96W + E239C + Q249R + G266D EISPPRRP (SEQ ID NO. 23) + G91A + D96W + E239C + Q249R + G266S E1SPPRRP (SEQ ID NO. 23) + G91A + D96W + E239C + Q249R + G263E + G266S + 270A E1SPPRRP (SEQ ID NO. 23) + G91A + D96W + E239C + Q249R + L264P + G266S E1SPPRRP (SEQ ID NO. 23) + G91A + D96W + E239C + Q249R + P2561 + G266D E1SPPRRP (SEQ ID NO. 23) + G91A + D96W + E239C + Q249R + 02660 + T267P + L269stop E1A + G91A + D96W + E99K + Q249R + G266S + T267S E1SPPCGRRP (SEQ ID NO. 25) + G91A + D96W + E239C + Q249R + P256T + G266S E1SPPCGRRP (SEQ ID NO. 25) + E239C + Q249R + P256T + G266S + T267A E1SPPCGRRP (SEQ ID NO. 25) + E2390 + Q249R + G266D E1SPPCGRRP (SEQ ID NO. 25) + G91A + D96W + E239C + Q249R + G266D E1SPPRRP (SEQ ID NO. 23) + D96S + E239C + Q249R + G266D L259S G266D G91A + 096W + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F (+274S) G266E G263A + G266A E1SPCRPRP (SEQ ID NO. 26) + E239C + Q249R + G266A E1SPCRPRP (SEQ ID NO. 26) + E239C + Q249R + G266S D96S + G266A D96S + G266S D96S + G266W E1SPPCGRRP (SEQ ID NO. 23) + D96S + E239C + Q249R + G263D + L264I + I265N + G266E + T267GS E1SPPCGRRP (SEQ ID NO. 23) + D96S + E239C + Q249R + L264I + I265N + G266T + T267GL D96F + G266A D96F + G266S E1SPPCGRRP (SEQ ID NO. 23) + E99N + E239C + Q249R + G266A E1SPPCGRRP (SEQ ID NO. 23) + D96S + E239C + Q249R + G266A E1SPPCGRRP (SEQ ID NO. 23) + D96S + E239C + Q249R + G266S E1SPPCGRRP (SEQ ID NO. 23) + D96S + E239C + Q249R + G263F + L264A + G266S + T267E V60G + D62A + S83T + R84K + D96W + G266D V60G + D62A + S83T + D96W + G266D V60G + D62A + S83T + D96W + G266W L259I L259N D96W + G263Q + L264A + I265T + G266D + T267A

In the table above, (+274S) indicates that the presence of this amino acid residue at the C-terminal is uncertain. For one such variant, it was found that only a minor fraction contained this residue

Several of the above variants had a higher ratio of phospholipase (PHLU) to lipase (LU) than a prior-art enzyme from F. oxysporum known to have both lipase and phospholipase activity.

For some of the above variants, the pH optimum for lipase and phospholipase was determined by using the LU and PHLU methods at various pH values. The results showed that the pH optimum phospholipase activity was in the range 4-6. The optimum for lipase activity varied from about pH 6 to about pH 10.

8 variants listed in Example 5 were analyzed for phospholipase activity by the mono layer assay described above at pH 5 and 9. The results showed that all the variants have phospholipase activity at pH 5 and 9, whereas the parent lipase (Humicola lanuginosa lipase) showed no activity at pH 5 or 9. Depending on the variant, the activity at pH 5 was higher or lower than at pH 9.

A prior-art variant of Humicola lanuginosa lipase was found to have no phospholipase activity at pH 5: SPIRR+N94K+F95L+D96H+N101S+F181L+D234Y+I252L+P256T+G263A+L264Q.

The following variants of the parent lipase from Humicola lanuginosa may also have phospholipase activity:

D62A + S83T + D96W + G266S G91A + D96W + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96L + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96N + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96A + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96E + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96S + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96R + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96G + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96Q + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96F + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96W + E99K + G263Q + L264A + I265T + G266S + T267A + L269N + 270A + 271G + 272G + 273F + 2743 G91A + D96F + E99K + G263Q + L264A + I265T + G2663 + T267A + L269N + 270A + 271G + 272G + 273F + 274S +  R84W + G91A + D96W + E99K + G263Q + L264A + I265T + G266S + T267A + L269N + 270A + 271G + 272G + 273F + 274S R84W + G91A + D96F + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S + R84W + G91A + D96F + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96F + G263Q + L264A + I265T + G2663 + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96W + G263Q + L264A + I265T + G266S + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96F + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96W + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S

Example 6 Variants of Rhizomucor Lipase with Phospholipase Activity

The following two variants of the parent lipase from Rhizomucor miehei were prepared and tested for phospholipase activity as described above. The variants were found to have phospholipase activity, where as the parent had no phospholipase activity by the same method.

G266N

G266V

Example 7 Variants of Humicola Lipase with Increased Specificity for Long-Chain Fatty Acids

Variants of the parent lipase from Humicola lanuginosa were prepared and tested for their hydrolytic activity on two triglyceride substrates with different chain length: tributyrin (C_(4:0)) and triolein (C_(18:1)). The tests were done at pH 9 by the LU and SLU methods described above. The following variants were found to have a higher ratio of triolein activity to tributyrin activity than the parent enzyme (Humicola lanuginosa lipase):

E1SPIRPRP (SEQ ID NO. 22) + G91A + D96N + E99K + Q249R E1SPCRPRP (SEQ ID NO. 26) + S83T + N94K + D96L + E239C + Q249R G266D E1SPIRPRP (SEQ ID NO. 22) + D62A + E99K + Q249R E1SPIRPRP (SEQ ID NO. 22) + D62G + E99K + Q249R E1SPIRPRP (SEQ ID NO. 22) + D62V + E99K + Q249R E1SPIRPRP (SEQ ID NO. 22) + R84W + E99K + Q249R E1SPIRPRP (SEQ ID NO. 22) + R84K + E99K + Q249R E1SPIRPRP (SEQ ID NO. 22) + K98D + E99K + Q249R E1SPIRPRP (SEQ ID NO. 22) + E99K + Q249R + 270PGLPFKRV E1SPPCGRRP (SEQ ID NO. 25) + E99N + N101S + T231K + R232G + D234G + E239C + Q249R E1SPIRPRP (SEQ ID NO. 22) + E99K + Q249R + 270PWPARLGRL L93K + D96G G91A + D96W + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F (+274S) E1SPCRPRP + V60G + E99N + S119G + R209P + E239C + Q249R G266A G266E G266V G263Q + L264A + I265T + G266D + T267A G266L G263A + G266A E1SPCRPRP (SEQ ID NO. 26) + E239C + Q249R + G266A E1SPCRPRP (SEQ ID NO. 26) + E239C + Q249R + G266S D96S + G266A D96S + G266S D96S + G266W L264I + I265N + G266T + T267GL E1SPPCGRRP (SEQ ID NO. 25) + D96S + E239C + Q249R + L264I + I265N + G266T + T267GL D96F + G266A D96F + G266S E1SPPCGRRP (SEQ ID NO. 25) + E99N + E239C + Q249R + G266A E1SPPCGRRP (SEQ ID NO. 25) + D96S + E239C + Q249R + G266A E1SPPCGRRP (SEQ ID NO. 25) + D96S + E239C + Q249R + G266S D62A + S83T E1SPPCGRRP (SEQ ID NO. 25) + K98D + E99N + E239C + Q249R T231R + N233R + 270CP E1SPPCGRRP (SEQ ID NO. 25) + E99N + E239C + Q249R + 270MD E1SPPCGRRP (SEQ ID NO. 25) + D62A + S83T + E99N + E239C + Q249R D62A + S83T + G91A + E99K + T231R + N233R + Q249R V60G + D62A + S83T + R84K + D96W + G266D L259N L259R L259M L259Q SPPCGRRP(-E) (SEQ ID NO. 25) + R84W + E99N + N101S + E2390 + Q249R R84W + G91A + E99K + T231R + N233R + Q249R Y21I Y21V SPIRPRP(-E) (SEQ ID NO. 22) + R84L + E99K + Q249R Y21C SPIRPRP(-E) (SEQ ID NO. 22) + D62 + E99K + Q249R D96W + G263Q + L264A + I265T + G266D + T267A + L269N + A270 + G271 + G272 + F273 + S274. G91A + D96W + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S

The following variants of the parent lipase from Humicola lanuginosa may also have an increased specificity for long-chain fatty acids:

SPIRPRP(-E) (SEQ ID NO. 22) + V60R + D62V + L93K + E99K + Q249R SPIRPRP(-E) (SEQ ID NO. 22) + D62V + E99K + Q249R SPIRPRP(-E) (SEQ ID NO. 22) + E99K + Q249R + P256D SPIRPRP(-E) (SEQ ID NO. 22) + D62V + E99K + Q249R + P256D SPIRPRP(-E) (SEQ ID NO. 22) + D62V + E99K + Q249R + P256S G91A + D96W + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96L + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96N + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96A + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96E + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96S + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96R + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96G + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96Q + E99K + G263Q + L264A + I265T + G266S + T267A + L269N + 270A + 2710 + 272G + 273F + 274S G91A + D96F + E99K + G263Q + L264A + I265T + G266S + T267A + L269N + 270A + 271G + 272G + 273F + 274S G91A + D96W + E99K + G263Q + L264A + I265T + G266S + T267A + L269N + 270A + 2710 + 272G + 273F + 274S G91A + D96F + E99K + G263Q + L264A + I265T + G266S + T267A + L269N + 270A + 271G + 272G + 273F + 274S R84W + G91A + D96W + E99K + G263Q + L264A + I265T + G266S + T267A + L269N + 270A + 271G + 272G + 273F + 274S R84W + G91A + D96F + E99K + G263Q + L264A + I265T + G266S + T267A + L269N + 270A + 271G + 272G + 273F + 274S R84W + G91A + D96F + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S SPPCGRRP(-E) (SEQ ID NO. 25) + V60G + D62E + S83T + R84K + E99N + N101S + E239C + Q249R V60G + D62E + S83T + R84K + G91A + E99K + T231R + N233R + Q249R

Example 8 Variants of Fusarium Lipase with Increased Specificity for Long-Chain Fatty Acids

Variants of the parent lipase from Fusarium oxysporum were prepared and tested as in the previous example. The following variants were found to have a higher ratio of triolein activity to tributyrin activity than the parent enzyme:

-   -   Y23S     -   Y260L

The following variants of the parent lipase from Fusarium oxysporum may also have an increased specificity for long-chain fatty acids:

R80H+S82T

S82T+A129T

Example 9 Variants of Rhizomucor Lipase with Increased Specificity for Long-Chain Fatty Acids

The following variants of the parent lipase from Rhizomucor miehei may have an increased specificity for long-chain fatty acids:

Y260W

Y28L

Y28C+H217N

Example 10 Variants of Humicola Lipase with Increased Specificity for Short-Chain Fatty Acids

Variants of the parent lipase from Humicola lanuginosa were prepared and tested as in the previous example. The following variants were found to have a higher ratio of tributyrin activity to triolein activity (a lower SLU/LU ratio) than the parent enzyme:

SPIRPRP(-E) (SEQ ID NO. 22) + E99K + R195Q + R209E + Q249R N101R + R195Q + R209E + L259S + Y261D N101R + R195Q + R209E + L259S N101R + L259S + Y2610 N101R + L259S Y261D L259S SPIRPRP(-E) (SEQ ID NO. 22) + E99K + N101R + Q249R G263D + L2641 + I265N + G266E + T267GS Y261I D234R Y261K

The following variants of the parent lipase from Humicola lanuginosa may also have a higher ratio of tributyrin activity to triolein activity:

N101R, R195Q, R209E, L259S, Y261D N101R, R195Q, R209E, L259S N101R, L259S, Y261D N101R, L259S

Example 11 Variants of Fusarium Lipase with Increased Specificity for Short-Chain Fatty Acids

Variants of the parent lipase from Fusarium oxysporum were prepared and tested as in the previous example. The following variants were found to have a higher ratio of tributyrin activity to triolein activity than the parent enzyme:

Y23W

Y260D

Y260R

Y260C

Y260N

Example 12 Variants of Rhizomucor Lipase with Increased Specificity for Short-Chain Fatty Acids

The following variants of the parent lipase from Rhizomucor miehei may have an increased specificity for short-chain fatty acids:

Y260C

Y260G

Y260V

Example 13 Variants of Humicola Lipase with DGDGase Activity

Variants of the parent lipase from Humicola lanuginosa were prepared, and the hydrolytic activity towards DGDG (di-galactosyl-di-glyceride) was determined as described above. The following variants were found to have DGDGase activity, whereas the parent lipase gave a negative result.

D96W + G263Q + L264A + I265T + G266D + T267A G263Q + L264A + I265T + G266D + T267A D96W + G263Q + L264A + I265T + G266D + T267A + L269N + 270AGGFS G91A + D96W + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270AGGFS D96F + G266S

Example 14 Variants of Humicola Lipase with Increased pH Optimum

Variants of the parent lipase from Humicola lanuginosa were prepared, and the lipase activity was measured by the LU method at pH 7 and 9. The following variants were found to have a higher ratio of activity at pH 9 to activity at pH 7 than the parent lipase:

R84L

R84W

Y21I

Y21V

Y261I

Example 15 Variants of Humicola Lipase with Decreased pH Optimum

Variants of the parent lipase from Humicola lanuginosa were prepared, and the lipase activity was measured by the LU method at pH 7 and 9. The following variants were found to have a lower ratio of activity at pH 9 to activity at pH 7 than the parent lipase:

Y261D

G266D/E

Y261W

Example 16 Use of Humicola Lipase Variants in Degumming of Vegetable Oil

Rapeseed oil was treated with two variants of the lipase from Humicola lanuginosa, essentially as described in Example 6 of WO 98/18912 (Novo Nordisk).

One variant was tested at an enzyme dosage of 0.6 mg of enzyme protein per kg of oil. Results of tests at various pH and temperatures showed optimum performance at pH 5.7, 35-45° C., where a final P content of 4 ppm was reached. A separate experiment at 45° C., pH 6 showed that a final P content of 4 ppm could be reached at an enzyme dosage as low as 0.15 mg/kg.

A similar experiment with another Humicola lanuginosa lipase variant showed optimum performance at 40° C., pH 5.0-5.5. The enzyme dosage was 0.3 mg/kg.

A degumming experiment was made with a third Humicola lanuginosa lipase variant, using rape seed oil at 45° C., pH 5, 1.8 mg enzyme/kg oil. For comparison, a similar experiment was made with the parent lipase (Humicola lanuginosa lipase) at 18 mg/kg. The results showed that good degumming (<10 ppm residual P content) was obtained in 3.4 hours with the variant.

The parent lipase (Humicola lanuginosa lipase) was found to have very little degumming effect, even at 10 times higher enzyme dosage.

Example 17 Use of Lipase Variants in Baking

A variant of the lipase from Humicola lanuginosa was evaluated in baking tests as follows.

Doughs were prepared from Meneba flour according to the European straight dough method (ABF-SP-1201.01) with 40 ppm of ascorbic acid. Various combinations of additives at the following dosages were used: the lipase variant at 0, 0.25, 0.5 or 1.5 mg/kg; phospholipid (lecithin) at 0 or 10 g/kg; and endo-amylase at 0 or 750 MANU/kg.

The endo-amylase was maltogenic amylase from B. stearothermophilus (tradename Novamyl®). One MANU (Maltogenic Amylase Novo Unit) is defined as the amount of enzyme required to release one mol of maltose per minute at a concentration of 10 mg of maltotriose substrate per ml of 0.1 M citrate buffer, pH 5.0 at 37 C for 30 minutes.

After baking, the loaves were cooled, and the loaf volume, crumb firmness and softness were evaluated after approximately 2 hours. The evaluation was repeated after 1, 3 and 7 days storage at 22° C. wrapped in double plastic bags.

Firmness of crumb was measured using a texture analyzer TA-XT2 from Stable Micro Systems (probe diameter 40 mm).

Softness in gram was measured as the force needed to press a probe 6.25 mm into a crumb of a 25 mm thick slice of bread (25% penetration).

The results showed that the addition of 1.5 mg of the variant increased the loaf volume. The results for firmness and elasticity show that the variant gives significantly softer crumb and significantly better elasticity from day 0 to day 7.

Example 18 Use of Lipase Variants for Dough Stability in Baking

A variant of the Humicola lanuginosa lipase was evaluated in a baking trial to evaluate its tolerance towards extended proofing of the dough.

Doughs were prepared from Pelikan flour according to the European straight dough method (347-SP-1217) with 30 ppm ascorbic acid, fungal-amylase (10 FAU of Fungamyl), and pentosanase (100 FXU of Pentopan Mono). Dosages of 0.2, 0.4, and 0.6 mg enzyme protein/kg flour of the variant were compared with 1000 LU of the parent lipase.

The doughs were made into rolls. Half of the rolls were proofed for 45 minutes (normal proofing) and the other half for 70 minutes (over proofing).

After baking the bread was cooled, and the volume and the standing of the rolls were evaluated after approximately 2 hours. The standing is a measure of the shape of the rolls and is defined as the height of 10 rolls divided by the width of 10 rolls, which means that nice round loaves have a high standing value, whereas flat rolls have a low standing value.

The results showed that at normal proofing time the volume of 0.4 and 0.6 mg of the variant were better than that of the parent lipase, and the standing of the rolls were better for the variant at all dosages than for the parent lipase. When the rolls were over proofed, both volume and standing was better for the variant at all dosages than for the parent lipase.

Example 19 Effect of Lipase Variants on Off-Odor Development

The development of off-odor from lipases with different chain-length specificity was evaluated in whole milk. The developed butyric acid/sour odor was evaluated by sniffing the samples after heating.

25 ml whole milk was placed in 100 ml blue cap flasks (with caps) in a 32° C. water bath. Of each of the lipases listed below, 0.2 mg enzyme protein per litre milk was added to the flasks. The temperature was raised to 45° C., and evaluation took place after 15 and 105 minutes.

The lipases tested were Humicola lanuginosa lipase and variants thereof. For each lipase, the chain-length specificity is expressed as the ratio of activities on triolein (SLU) and tributyrin (LU).

Three persons evaluated the samples and agreed on the ranking shown below

+ Detectable smell

++ Clear and characteristic butyric acid and/or sour odor

+++ Strong butyric acid and/or sour odor

Three variants of Humicola lanuginosa lipase having a higher SLU/LU ratio than Humicola lanuginosa lipase were found to have less malodor than the parent lipase.

Example 20 Effect of Lipase Variants on Malodour on Textile after Washing

Soiling:

Cotton textile was soiled with a dairy product as described here. 50 mg of butter was applied over an area of approximately 30 cm² in an even spot. The soiled textile was aged for 24 hours at ambient conditions.

Washing Procedure:

Washing of the soiled textile was done in a Terg-O-tometer using a commercial detergent (5 g/l) with and without lipase (1250 and 5000 LU/l). The washing was done at 30° C. for 20 min at 100 rpm. After washing the swatches were left overnight to dry at ambient conditions.

Sensory Analysis:

Next day, the malodour was assessed by a sensory panel consisting of at least 10 trained assessors. Samples were kept in tight glass jars and left at least 30 minutes between every evaluation for accumulation of malodour. Swatches were taken out and the malodour assessed on the textile. The butyric acid malodour was scored according to the scale below. As a reference the sample washed without lipase was used.

0. Fainter smell than reference

1. Same as reference

2. Slightly stronger than reference

3. Definitely stronger than reference

4. Stronger than 3.

Variants of Humicola lanuginosa lipase with an increased ratio of triolein/tributyrin activities (increased SLU/LU ratio) were found to give a fainter smell from butter stains than the parent enzyme (Humicola lanuginosa lipase). A separate washing experiment showed that the variants, like the parent enzyme, were effective in the removal of lard stains.

Alternative Methods

The intensity of butyric acid from dairy stains on fabric can also be evaluated by instrumental analysis:

1. By Head Space Gas Chromatography, or

2. By extraction of the odours from fabric followed by Gas Chromatography

Example 21 Effect of Lipase Variants on Odour of Bread Baked with Butter

Six variants of the lipase from Humicola lanuginosa were prepared and were evaluated in bread baked by the European straight dough procedure (347-SP-1217) with addition of 3% butter. 0.2 mg enzyme protein/kg flour was used for each of the variants.

The chain-length specificity of the variants was also determined by measuring the triolein/tributyrin activity ratio (SLU/LU described above). The parent lipase from Humicola lanuginosa and a prior-art lipase with phospholipase activity from Fusarium oxysporum were also tested for comparison.

The results are summarized below:

SLU/LU Rating Variants 2.7 (+) of the 3 no effect invention 7 no effect 28 no effect 70 no effect Parent lipase 1.2 ++ Prior-art lipase 1.1 +++ Control (no lipase) — no effect

The results indicate that that lipase variants with a SLU/LU ratio at 3 or above (i.e. a high specificity for long-chain fatty acids) give no unpleasant odour in bread baking even with butter in the recipe. 

1. A method of producing a lipolytic enzyme variant comprising: a) selecting a substrate triglyceride comprising a glycerol part having an sn2 position, and an ester bond of interest, b) selecting a parent lipolytic enzyme having an alcohol binding site capable of binding the glycerol part of the substrate triglyceride, c) selecting at least one amino acid residue in the parent lipolytic enzyme which comprises at least one atom within 10 Å of the C atom at the sn2 position of the glycerol part of the substrate triglyceride, d) making an alteration of the at least one amino acid residue of the parent lipolytic enzyme to provide a variant, e) preparing the variant of step d), f) testing the activity of the variant on the selected ester bond, g) selecting a variant having an altered activity on the selected ester bond, and h) producing the selected variant; wherein the parent lipolytic enzyme is a fungal lipolytic enzyme having at least 95% homology with SEQ ID NO:32 or having at least 95% homology with SEQ ID NO:30.
 2. The method of claim 1, wherein the parent lipolytic enzyme is a lipase.
 3. The method of claim 1, wherein the parent lipolytic enzyme is a Humicola lipolytic enzyme.
 4. The method of claim 1, wherein the parent lipolytic enzyme is a H. lanuginosa lipolytic enzyme.
 5. The method of claim 1, wherein the parent lipolytic enzyme is a Fusarium solani pisi lipolytic enzyme.
 6. The method of claim 1, wherein the parent lipolytic enzyme is a Fusarium oxysporum lipolytic enzyme.
 7. The method of claim 1, wherein the parent lipolytic enzyme is a Rhizomucor miehei lipolytic enzyme.
 8. The method of claim 1, wherein the parent lipolytic enzyme is a lipase having phospholipase activity corresponding to a ratio of phospholipase activity to lipase activity below 0.1 PHLU/LU.
 9. The method of claim 1, wherein the parent lipolytic enzyme is a lipase having phospholipase activity corresponding to a ratio of phospholipase activity to lipase activity below 50 PHLU/mg.
 10. The method of claim 1, wherein step c) comprises selecting at least one amino acid residue in the parent lipolytic enzyme which comprises at least one atom within 8 Å of the C atom at the sn2 position of the glycerol part of the substrate triglyceride.
 11. The method of claim 1, wherein step c) comprises selecting at least one amino acid residue in the parent lipolytic enzyme which comprises at least one atom within 6 Å of the C atom at the sn2 position of the glycerol part of the substrate triglyceride.
 12. The method of claim 1, wherein the method comprises making no more than 10 alterations in the parent lipolytic enzyme.
 13. The method of claim 1, wherein the method comprises making no more than 6 alterations in the parent lipolytic enzyme.
 14. The method of claim 1, wherein the method comprises making no more than 5 alterations in the parent lipolytic enzyme.
 15. The method of claim 1, wherein the method comprises making no more than 4 alterations in the parent lipolytic enzyme.
 16. The method of claim 1, wherein the method comprises making no more than 3 alterations in the parent lipolytic enzyme.
 17. The method of claim 1, wherein the method comprises making no more than 2 alterations in the parent lipolytic enzyme.
 18. The method of claim 1, wherein the method comprises making no more than 1 alteration in the parent lipolytic enzyme. 